Polyoma virus assembly occurs by sequential addition of capsid proteins to the viral minichromosome to form a complete T=7d icosahedral virion. The overall objective of this proposal is a structure-function analysis of the polyma capsid proteins with respect to three aspects of virion assembly: 1) the formation of capsomeres and capsids, 2) their interaction with the viral minichromosome, and 3) their transport to the site of virus assembly in the nucleus. The major capsid protein VP1, purified after high level espression in E. coli, will be studied by electron microscopy and physical chemical methods, and recombinant VP1 capsomeres will be crystallized for X-ray diffraction analysis. Protein domains of VP1 responsible for capsomere and capsid assembly will be investigated using temperature-sensitive viruses carrying mutations in VP1, and by altering the VP1 gene in vitro using recombinant DNA techniques. The ability of the recombinant VP1 protein to assemble spontaneously into capsids will be used to encapsidate viral DNA and minichromosomes in vitro. The protein domain of VP1 which facilitates its nuclear transport will be studied using VP1-beta-galactosidase fusion protein expression in yeast (S. cerevisiae) and by microinjection of recombinant VP1 capsomeres into Xenopus oocytes. The expression in E. coli of the genes for the minor capsid proteins VP2/VP3 will enable a similar structure-function analysis of these proteins. As a preliminary step towards the long range goal of studying virus-receptor interactions, antibodies against the putative mouse cell receptor protein for polyoma will be used to clone the polyoma receptor gene. We anticipate that our studies will have significance for normal cell functions such as the assembly of macromolecules and the subcellular localization of proteins. In addition, a more complete understanding of virus assembly will be relevant to elucidating molecular mechanisms of viral pathogenesis, and to designing and understanding anti-viral chemotherapy.